Modular multiple fluid sample preparation assembly

ABSTRACT

An apparatus for collecting and testing multiple biological markers comprising a tubular compartmentalized container holding covalently bound antigen beads and correlating anti-antibody beads contained in separated compartments. The biological fluid, namely urine, is collected in the tubular container and is forced to flow through the separated compartment of the compartmentalized container so that predetermined ligands become attached to the bead ligands to obtain a plurality of biological markers.

RELATED CASES

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 07/369,610 filed June 21, 1989.

BACKGROUND OF THE INVENTION

The present invention is directed to medical and laboratory specimencollecting and testing equipment, and more specifically to an apparatusfor detecting the presence of a plurality of specific antigens inbiological fluids.

It is generally necessary in diagnosing and testing for many diseases tocollect biological fluids from a patient, e.g., sputum, blood, pleuraland peritoneal cavity fluids, urine, etc. for analysis. It is importantduring the collection handling of biological fluid specimens that thepotential of specimen contamination and the spread of any infection fromthe specimen be minimized. In addition there is also the potential forspecimen damage during the collection and/or shipment process as well asthe potential for destruction of certain components of the specimenbecause the packaging does not screen particulants in the fluids orcollects and holds different fluid components which will negate the testresults or result in false data being obtained when the specimen istested.

It has been noted that one of the problems in collecting biologicalfluid specimens occurs not only during the collection of the specimensbut also in the transport or shipment of the specimens after collectionto the laboratory for analysis.

A typical specimen collecting apparatus is shown by U.S. Pat. No.4,741,346. This apparatus includes a base stand which supports thespecimen vial in an upright position. A funnel is inserted in the openend of the specimen vial and surrounds and encloses the upper portion ofthe vial. The base stand has an upwardly extending tubular wall which atleast partially surrounds the vial in connection with the cap and allowsthe user to remove the vial without touching the surface or coming incontact with the specimen. Examples of various types of liquidcontainers for collecting and transporting urine are shown by U.S. Pat.Nos. 3,777,739; 3,881,465; 4,042,337; 4,084,937; 4,244,920; 4,492,258and 4,700,714.

One such specimen collection device shown by U.S. Pat. No. 4,040,791discloses a collection receptacle having a nipple upon which is mounteda specimen container which receives a predetermined amount of thespecimen in a sealed condition. The specimen container is provided withan integally formed cap which is placed over the opening in which thecollector nipple is inserted. U.S. Pat. No. 4,557,274 discloses amidstream urine collector having a funnel which transmits urine into acup member which is covered by a membrane cover.

A combined strip testing device and collection apparatus is shown byU.S. Pat. No. 4,473,530 and is directed to an apparatus which integratestesting and collection by having chemical reagent test strips presentwithin the tube together with specific gravity reading means allowingimmediate testing of the urine. U.S. Pat. No. 4,573,983is directedtowards a liquid collection system having an antiseptic member on thedischarge section which uses a filter of air and bacteria imperviousmaterial to filter the urine.

It is therefore desirable to provide an easy to handle apparatus whichuses a fluid sample such as urine and separates various antibodies fromurine so that testing can be performed quickly and accurately withminimum time.

It is thus an object of the invention, particularly where antigens arebeing removed from the body fluids for a variety of tests to detect andvisually indicate specific antigens in the body fluid samples.Previously such testing has been accomplished by a series of testsinvolving a number of different containers and expensive laboratoryequipment. Mass testing using such a series of tests is expensive, timeconsuming, and often unsatisfactory.

BRIEF SUMMARY OF THE INVENTION

The invention is directed toward a multiple antigen marker analysisdevice. This device is in the form of a modular sample container in thehousing, which can be washed or treated with biological fluids allowingthe fluid sample to be quickly analyzed. The antibodies (against theantigen) and antigens are covalently bound to beads contained inconcentric separated compartments formed in a single container. Theantibodies can be provided prelabelled with coloring reagents. Thetesting sample is added to the container where the respective antigenreacts with the respective antibody to form antigen-antibody complex. Ifthere is an absence of the antigen and/or antibody in the specimensample the antibody will remain unoccupied. The beads housing unitcontains three different sets of beads, one set with antigencovalently(irreversible) bound, one set with antibody covalently boundand the other set without antigen or antibody to act as a control.

In the accompanying drawings, there is shown an illustrative embodimentof the invention from which these and other of objectives, novelfeatures and advantages will be readily apparent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross sectional view of separated units of the samplecollection apparatus of the invention;

FIG. 2 is a cross sectional view of a cap for the shuttle storage unitof the sample collection apparatus of FIG. 1;

FIG. 3 is an exploded cross sectional view of the piston assembly andsample/test container of the shuttle apparatus which fits into thesample collection apparatus of FIG. 1;

FIG. 4 is a cross sectional view of a sample filtration purificationshuttle apparatus with direction of movement of the plunger shown byarrow D' and direction of flow of the fluid shown by arrows A;

FIG. 5 is an enlarged perspective view partially in cross section of thesample test container;

FIG. 6 is a view of the sample test container of FIG. 5 with labelledantigen and antibody beads positioned in the respective compartments;

FIG. 7 is a table showing representation of the interpretation of testresults; and

FIG. 8 is a top plan view of the top surface of the sample testcontainer showing different test zones.

DETAILED DESCRIPTION OF THE INVENTION

The preferred embodiment and best mode of the invention is seen in FIGS.1 through 6. The invention shown in the drawings comprises a modularfluid sample container with sample collection apparatus. While theinvention can be used for any body fluid such as sputum, blood, bodyfluids or urine, it is primarily designed for use in collectingurine/blood samples for use in testing for the presence of various kindsof cancer in the body.

As shown in FIGS. 1 and 3 a sample collection apparatus 20 isconstructed of polystyrene and comprises a tubular collection unit 22, ashuttle storage unit 30 and an associated cap member 40. The tubularcollection unit 22 is constructed with a tubular open ended cylindricalbody 24 having formed on one end an open flared end portion 26 and onthe other end a threaded portion 28. The flared end portion 26 has awide mouth to more easily receive body fluid which is loaded into theunit and also prevents entrapment of air between the bead container unit70 and the surface of the sample fluid. The shuttle storage unit 30 isremovably secured to the threaded portion 28. The shuttle storage unit30 comprises a cylindrical cup shaped body 32 with a stepped femalethreaded open end 34 and shoulder 35. The open end 34 has a greaterdiameter than the diameter of body 32. A stop member 36 comprised of acircular ring or integrally formed rib is formed or secured to the innersurface of the shuttle body 32 immediately below the shoulder 35 of thethreaded stepped open end 34. This stop member 36 serves as a stop for atransporter or shuttle assembly 50 shown in FIG. 3 which will bediscussed later on in the specification. The cap member 40 as shown inFIG. 2 is formed with a cylindrical body 42 having a lid or cover 44 andexternal threads 46 which mate with the female threaded end portion 34of the shuttle body 32. Thus the shuttle storage unit 30 and cap 40 canbe used to form a closed container holding the particulants, testingmaterial or fluid as desired. It should also be noted that a threaded orsnug fitting cap (not shown) can be placed over end portion 26 so thatthe sample collection apparatus can be closed.

The transporter assembly 50 as shown in FIG. 3 is designed to fit withincylindrical body 24 and slideably move along the interior wall surface25 in a sealed relationship and abut a shuttle sample container 70 fordeposit within the shuttle storage body 32. The transporter assembly 50is constructed of a transparent plastic comprising a hollow cylindricalpiston body 52 provided with a thumb cover 54 and a bottom end member55. An air release aperture 56 is formed in the piston body so thatthere is communication between the interior chamber 53 of the pistonbody into the outside atmosphere. The bottom end member 55 is porous andallows fluid flow therethrough into the interior chamber 53 as theexterior rigid surface engages the outer surface of the container 70.Mounted around the piston body in annular channels 61 and 63 cut intothe exterior surface of the body 52 are respectively an upper O-ring 62and lower O-ring 64. These O-rings slideably engage and form a fluidseal against the interior surface 25 of sample collection unit 22. Theshuttle sample container 70 is adapted to receive end member 55 withinthe upper inner wall surface of the container cylindrical body 72 andhas a diameter or size sufficient to allow it to be seated within theinterior 33 of shuttle storage unit 30.

The shuttle sample container 70 as clearly shown in FIG. 5 isconstructed with a cylindrical body 72 open at both ends and threaded toallow the mounting of a porous circular center top member 74 which isthreaded on the inside of the cylindrical body 72 and defines anaperture 83 which holds the outer cylinder 84. The outer cylinder 84 andassociated top cover 74 are preferably sonically welded to each otherbut may be joined together by adhesive or any means known in the art.

A second top cover member 78 is stacked on top of the initial top member74 and defines an aperture 79 which holds the first inner cylinder 80. Athird top cover member 82 is stacked on top of the second top covermember 78 and defines an aperture 75 which holds a central cylinder 76.A top cover 86 is stacked on top of the third top cover member 82 tocover the top of the cylinder body 72. These porous cover members aresecured to their respective cylinders as previously noted above and canbe threaded on the inside of the cylindrical body 72 or constructed witha male/female mating construction on their upper and lower surfaces suchas a tongue and groove which will allow the covers to be snappedtogether Alternately an outer cover (not shown) could be threadablymounted to the outside of the cylinder body 72 to hold the stacked covermembers in fixed relationship to each other. The concentric nature andspacing of the respective cylinders 76, 80, 84 and 72 forms chambers 77,81, 85 and 87. A circular disc membrane 100 preferably between 0.45-5.0microns in thickness is positioned on the outer surface of top cover 86.A circular porous bottom cover 90 is threaded on the inside of the otherend of cylinder body 72. The ends of cylinders 76, 80 and 84 abutagainst the upper surface of the bottom cover 90. Alternately the bottomcover 90 can be cut with circular grooves into which the ends of thecylinders 76, 80 and 84 fit or be provided with circular ribs into whichthe ends of the cylinders are seated and sealed. The body 72 cylinders76, 80 and 84, top cover 86 and bottom cover 90 define a plurality ofspaced chambers 77, 81, 85 and 87 which are filled with a predeterminedsequence of beads which are covalently bound to various antigen andantibodies which constitute the biological markers. Positioned insidethe central cylinder 77 is a test bead module 91 filled with unboundbeads which acts as a control. The adjacent bead module 92 is filledwith two sets of beads namely; antigen A, covalently bound beads 110 andanti-B antibody covalently bound beads 115. The adjacent outer beadmodule 94 is filled with two sets of beads namely; anti-B antibodycovalently bound beads 115 and anti-A antibody covalently bound beads120. The outer bead module 96 is filled with antigen B covalently boundbeads 125 and antigen A covalently bound beads 110. Thus it can be seenthat the beads are specifically combined to obtain a multiple markertest as shown in FIG. 8.

An O-ring sleeve 102 comprising a plurality of O-rings 104 is mounted tothe exterior surface of the cylinder 72 formed by the sides of both topand lower covers 86, 90 when the same are fully screwed onto the body ofthe container 72. While specific sample beads have been described it iswithin the present invention that the chambers of the container 70 maybe filled with sample beads of all forms and sizes which can bespecifically manufactured for high affinity chromatography.

The beads can also have high affinity chromatography. The chemical andmechanical stability of the support (resin) and its linkage to theligand (antigen) play a key role in affinity chromatography. Preferablyas has been previously described the module bead chambers hold highaffinity resin beads with specific antibodies or antigens immobilizedonto the solid phase resin (e.g. Actigel-ALD, Protein A, Protein G . . .etc.) so that antigens in the sample can bind to their specificantibodies, or alternately the antibody complexes can become bound whilepassing through the resin module and become immobilized as well. Theprinciple of affinity chromatography requires that a successfulseparation of a biospecific ligand is available and that it can bechemically immobilized to a chromatographic bed material, the matrix.Numbers of methods well known in the art have been used to couple orimmobilize the antigen to a variety of activated resins. Examples ofimmobilization techniques which exhibit variable linkage are thoseformed by the reaction of the reactive groups on the support with amino,thiol, hydroxyl, and carboxyl groups on the protein ligand. Theimmobilized ligand must also retain its specific binding affinity forthe substance of interest with the bound substances being selectivelydesorbed in an active form. The selection of the ligand is influenced bytwo factors. First, the ligand should exhibit specific and reversiblebinding affinity for the substance to be purified and secondly it shouldhave chemically modifiable groups which allow it to be attached to thematrix without destroying its binding activity. (Examples of such areProtein G Sepharose manufactured by Pharmacia, Hydrazide AvidGel Axmanufactured by BioProbe International, and Actigel-ALD manufactured bySterogene Bioseparation Inc.)

In the case of Actigel-ALD a monomonic, stable, low reactively aldehydegroup is introcuded to the support through 5 atoms hydrophilic spacerarm. This establishes well defined, stable secondary amine linkageswithout side reaction or the release of hydrophobic or reactive leavinggroups. Another advantage to the use of Actigel-ALD is that it does notcross link proteins therfore allowing proteins to retain highbioactivity after their immobilization. Actigel-ALO SUPER FLOW alsoavailable from Sterogene Bioseparation Inc. permits a linear flow rateof up to 3000 cm/h which would fit nicely with the flow rates in thedevice (approx 10-100 cm/min).

Visual affinity chromatography can be obtained through the use of colormarkers in the form of dye affinity chromatography product are availableand are manufactured by TosoHaas under the designation TSK-GEL TOYOPEARLDYE AFFINITY CHROMATOGRAPHY PRODUCTS made with TOYOPEARL HW-65 as thebase matrix and Cibacron Blue F3GA or reactive red 120 as the dyereagent. Blue-TOYOPEARL 650 and Red-TOYOPEARL 650 have differing proteinbinding capacities.

The shuttle resin/sample container 70 is pushed down until it entersinto the body cavity 33 at which time the lower O-ring 64 engages stoprib 36 thus seating the shuttle resin/sample container 70 in apredetermined position so that it cannot break or damage the shuttlestorage unit 30. When the shuttle resin/sample container 70 is seated inthe shuttle storage unit 30, the upper "O" ring 62 becomes engaged withthe inner surface 25 of cylindrical body 24. This will discontinue thecommunication between the body cavity 53 and the atmosphere through theair release aperture 56. Consequently the fluid entering the body cavity53 will be trapped inside it even after removal of the shuttle storageunit 30 together with the seated resin/sample container 70.

The resin bead material with matrix and primary ligand (in this caseantigen and antibody) having had flow contact with the fluid, namelyurine, captures through antigen-antibody reaction or immune reaction thespecific component of the fluid which is to be tested; and shows themultiple antigen marker status as per FIGS. 7 and 8. The primaryantibodies against the antigens can be provided with prelabelledcoloring reagents. A buffering reagent is also provided with the samplecontainer to optimize the antigen antibody reaction (e.g. pH 8) sincethe urine pH is usually acidic. The testing sample is added to thecontainer where the antigen reacts with the antibody to formantigen-antibody complexes. If there is an absence of the antigen in thespecimen sample the antibody will remain unoccupied and will react withthe antigen immobilized on beads. On the other hand, if the antigen ispresent, anitgen-antibody complexes will be formed. The beads housingcontainer unit contains five different sets of beads having the ligandcovalently bound thereto, one set with antigen A, and one set antigen B,one set with anti-A antibody and one set with anti-B antibody and thefifth set without any legand to act as a control. On the top of thebeads housing, there is a circular (disk) high affinity membrane withimmobilized secondary antibody against the primary antibody species. Theupper surface of the disk membrane 100 provides the surface upon whichthe primary antibody and/or antigen-antibody complexes are captured bythe secondary antibody immobilized on the top surface of the membraneand the test result is visualized.

The shuttle storage unit 30 is unscrewed from the tubular collectionunit 22 with the shuttle resin/sample container 70 contained therein,the transporter assembly 50 remaining with the tubular collection unit22 and cap 40 is screwed on threaded end 34 to keep the sample in asecured contained condition after adding the proper preservatives foranalysis of the particulate matter or for testing by pouring colordeveloping solution to visualize the prelabeled antibody captured on thetop surface of the circular disk with the immobilized capturingsecondary antibody.

In the foregoing description, the invention has been described withreference to a particular preferred embodiment, although it is to beunderstood that specific details shown are merely illustrative, and theinvention may be carried out in other ways without departing from thetrue spirit and scope of the following claims:

What is claimed is:
 1. An apparatus for collecting molecular samples from biological fluids to obtain multiple markers comprising a first container means; a sample collection storage unit removably mounted to said first container means, said sample collection storage unit comprising a container divided into plurality of bead containing compartments, control bead means positioned in one said compartments, at least two different antigen bead means located in at least another of said compartments; and at least two different anti-antibody bead means corresponding to said antigen bead means located in at least one other of said compartments.
 2. An apparatus as claimed in claim 1 wherein said antigen bead means comprise beads having antigen A covalently bound thereto and beads having antigen B covalently bound thereto.
 3. An apparatus as claimed in claim 1 wherein said anti-antibody bead means are anti-A antibody beads and anti-B antibody beads.
 4. An apparatus as claimed in claim 1 wherein said plurality of compartments comprise at least 3 compartments; one of said compartment containing antigen A beads and anti-B antibody beads, another of said compartments containing anti-A antibody beads and anti-B antibody beads, and one compartment containing anti-B antibody and antigen B beads.
 5. An apparatus as claimed in claim 1 wherein said plurality of compartments comprise at least 4 compartments; one of said compartments containing antigen A beads and anti-B antibody beads, another of said compartments containing anti-A antibody beads and anti-B antibody beads, another compartment containing anti-B antibody and antigen B beads, and yet another compartment containing antigen A beads and antigen B beads.
 6. An apparatus as claimed in claim 1 wherein said container means comprises an outer body, a plurality of concentric tubular cases and a top cover means and a bottom cover means.
 7. An apparatus as claimed in claim 1 wherein said container means comprises a cylindrical housing, cover plate means covering each end of said cylindrical housing, each cover plate means allowing flow of fluids therethrough, and membrane means extending across one end of the cylindrical housing, said membrane means being provided with immobilized secondary antibody against the primary antibody species.
 8. An apparatus as claimed in claim 7 wherein said membrane means comprises a membrane ranging from 0.45 to 5.0 microns in thickness.
 9. An apparatus for collecting multiple markers from biological fluids comprising a tubular container; a specimen collection unit mounted in said tubular container, said specimen collection unit comprising a housing constructed of concentric tubular walls forming spaced chambers therebetween, bead means comprising at least two different covalently bound antigen beads positioned in at least one of said chambers, control bead means without antigen located in another of said chambers; and bead means comprising at least two different covalently bound primary antibodies positioned in at least another chamber and top and bottom cover means on each end of said tubular container.
 10. An apparatus as claimed in claim 9 wherein "O" ring means are mounted to the outside of said tubular housing.
 11. An apparatus as claimed in claim 10 wherein said "O" ring means comprises a sleeve, said specimen collection unit defining at least four concentric separate bead containing chambers.
 12. A container apparatus for collecting a plularity of predetermined markers from biological fluids comprising a cylindrical housing with end means allowing fluid flow therethrough, means dividing said housing into at least four separated isolated chambers, each chamber containing a plurality of beads provided with immobilized ligand means, one of said chambers holding ligand means comprising antigen A and/or antigen B beads and one of said chambers holding control beads, one of said chambers holding ligand means comprising primary anti-A antibody beads and/or anti-B antibody beads and a membrane positioned on one end of said housing with immobilized secondary antibodies affixed thereto.
 13. An apparatus for collecting multiple markers from biological fluids comprising a housing removably mounted to a pump means, a biological marker collection means mounted in said housing, said biological marker collection means comprising a plurality of stackable chamber assemblies mounted in said housing to form a plurality of distinct separated chambers, each chamber assembly comprising a porous support member defining a throughgoing aperture, a hollow conduit member mounted to said support member in alignment with said support member aperture to allow fluid flow therethrough, said chamber assemblies when mounted in said housing forming a plurality of distinct separated chambers which separate biological fluid within said housing while allowing biological fluid to flow therethrough, ligand means contained in at least two chambers formed by said chamber assemblies, said ligand means being specifically biologically configured to capture different ligand markers from biological fluid transported through said chambers by said pump means.
 14. An apparatus for collecting multiple markers from biological fluids as claimed in claim 13 where said conduit members are cylindrical and are axially aligned with the aperture defined by said porous support member.
 15. An apparatus for collecting multiple markers from biological fluids comprising a cylindrical housing open at both ends, said housing being adapted to be removably mounted to a pump means, a specimen collection means mounted in said housing, said specimen collection means comprising a plurality of stackable chamber assemblies removably mounted and concentrically positioned in said housing, each chamber assembly comprising a planar surfaced porous support member defining a throughgoing aperture, a hollow cylindrical conduit member mounted to said support member and extending therefrom, said conduit member being axially aligned with said support member aperture to allow fluid flow therethrough, and a porous cover member mounted to said housing and engaging the distal concentric positioned ends of said conduit members of said chamber assemblies, a circular membrane positioned on the outer surface of the outermost chamber assembly support member, said membrane being provided with an immobilized secondary antibody on its surface, said chamber assemblies when mounted to said housing forming a plurality of concentric separated chambers within said housing, ligand means contained in at least two chambers formed by said chamber assemblies, said ligand means being specifically biologically configured to capture different ligand markers from biological fluid which is transported through said housing by said pump means.
 16. An apparatus for collecting multiple markers from biological fluids as claimed in claim 15 where said membrane ranges between 0.45 and 5.0 microns in thickness.
 17. An apparatus for collecting multiple markers from biological fluids as claimed in claim 15 where said ligand means in at least two chambers comprise beads which are bound to predetermined various different antigen which constitute biological markers.
 18. An apparatus for collecting multiple markers from biological fluids as claimed in claim 15 where said ligand means in at least two chambers comprise beads which are bound to predetermined various different antibodies which constitute biological markers.
 19. An apparatus for collecting multiple markers from biological fluids as claimed in claim 15 where said ligand means at least two chambers comprise beads which are bound to predetermined various different antigens and antibodies which constitute biological markers.
 20. An apparatus for collecting multiple markers from biological fluids as claimed in claim 15 where said ligand means in one chamber comprises beads which are unbound which acts as a control. 